Alina Codita

Consistent behavioral phenotype differences between inbred mouse strains in the IntelliCage

The between‐laboratory effects on behavioral phenotypes and spatial learning performance of three strains of laboratory mice known for divergent behavioral phenotypes were evaluated in a fully balanced and synchronized study using a completely automated behavioral phenotyping device (IntelliCage). Activity pattern and spatial conditioning performance differed consistently between strains, i.e. exhibited no interaction with the between‐laboratory factor, whereas the gross laboratory effect showed up significantly in the majority of measures. It is argued that overall differences between laboratories may not realistically be preventable, as subtle differences in animal housing and treatment will not be controllable, in practice. However, consistency of strain (or treatment) effects appears to be far more important in behavioral and brain sciences than the absolute overall level of such measures. In this respect, basic behavioral and learning measures proved to be highly consistent in the IntelliCage, therefore providing a valid basis for meaningful research hypothesis testing. Also, potential heterogeneity of behavioral status because of environmental and social enrichment has no detectable negative effect on the consistency of strain effects. We suggest that the absence of human interference during behavioral testing is the most prominent advantage of the IntelliCage and suspect that this is likely responsible for the between‐laboratory consistency of findings, although we are aware that this ultimately needs direct testing.

Influence of environmental manipulation on exploratory behaviour in male BDNF knockout mice

It is widely accepted that brain derived neurotrophic factor (BDNF) plays a crucial role in mediating changes in learning and memory performance induced by environmental conditions. In order to ascertain whether BDNF modulates environmentally induced changes in exploratory behaviour, we examined mice carrying a deletion in one copy of the BDNF gene. Young heterozygous male BDNF knockout mice (BDNF+/-) and their wild-type (WT) controls were exposed to the enriched environment condition (EC) or the standard condition (SC) for 8 weeks. Exploratory behaviour was assessed in the open-field (OF) and hole-board (HB) test. Brains from EC and SC reared animals were processed for Golgi-Cox staining and the dendritic spine density in the dentate gyrus (DG) and CA1 hippocampal regions were examined. We found behavioural differences both due to the genetic modification and the environmental manipulation, with the BDNF+/- mice being more active in the OF whereas the EC mice had increased exploratory behaviour in the HB test. Environmental enrichment also led to an increase in dendritic spines in the hippocampal CA1 region and DG of the wild-type mice. This effect was also found in the enriched BDNF+/- mice, but was less pronounced. Our findings support the critical role of BDNF in behavioural and neural plasticity associated with environmental enrichment and suggest that besides maze learning performance, BDNF dependent mechanisms are also involved in other aspects of behaviour. Here we provide additional evidence that exploratory activity is influenced by BDNF.

Alterations in brain leptin signalling in spite of unchanged CSF leptin levels in Alzheimer's disease

Several studies support the relation between leptin and Alzheimers disease (AD). We show that leptin levels in CSF are unchanged as subjects progress to AD. However, in AD hippocampus, leptin signalling was decreased and leptin localization was shifted, being more abundant in reactive astrocytes and less in neurons. Similar translocation of leptin was found in brains from Tg2576 and apoE4 mice. Moreover, an enhancement of leptin receptors was found in hippocampus of young Tg2576 mice and in primary astrocytes and neurons treated with Aβ1‐42. In contrast, old Tg2576 mice showed decreased leptin receptors levels. Similar findings to those seen in Tg2576 mice were found in apoE4, but not in apoE3 mice. These results suggest that leptin levels are intact, but leptin signalling is impaired in AD. Thus, Aβ accumulation and apoE4 genotype result in a transient enhancement of leptin signalling that might lead to a leptin resistance state over time.

Impaired behavior of female tg-ArcSwe APP mice in the IntelliCage: A longitudinal study

Transgenic animals expressing mutant human amyloid precursor protein (APP) are used as models for Alzheimer disease (AD). Ideally, behavioral tests improve the predictive validity of studies on animals by mirroring the functional impact of AD-like neuropathology. Learning and memory studies in APP transgenic models have been difficult to replicate. Standardization of procedures, automatization or improved protocol design can improve reproducibility. Here the IntelliCage, an automated system, was used for behavioral testing of APP female transgenic mice with both the Arctic and Swedish mutations, the tg-ArcSwe model. Protocols covering exploration, operant learning, place learning and extinction of place preference as well as passive avoidance tests were used for longitudinal characterization of behavior. Differences in exploratory activity were significant at four months of age, when plaque-free tg-ArcSwe mice visited less frequently the IntelliCage corners and initially performed fewer visits with licks compared to non-tg animals, inside the new environment. Fourteen months old tg-ArcSwe mice required a longer time to re-habituate to the IntelliCages than non-tg mice. At both ages tg-ArcSwe mice perseverated in place preference extinction test. Fourteen months old tg-ArcSwe mice were impaired in hippocampus-dependent spatial passive avoidance learning. This deficit was found to inversely correlate to calbindin-D28k immunoreactivity in the polymorphic layer of the dentate gyrus. Reduced water intake and body weight were observed in 4 months old tg-ArcSwe animals. The body weight difference increased with age. Thus behavioral and metabolic changes in the tg-ArcSwe APP model were detected using the IntelliCage, a system which provides the opportunity for standardized automated longitudinal behavioral phenotyping.

Of mice and men : more neurobiology in dementia.

Purpose of review An increasing number of genetically modified mouse models are designed and used in the field of Alzheimer disease research. This review aims to offer a general view of the existing transgenic mouse lines and to discuss their relevance and limitations. Recent findings Potential therapeutic targets have been identified in rodent models of Alzheimer disease. Although important steps towards obtaining a safe vaccine to prevent amyloid plaque formation have been made, further evaluations and the use of intermediate models are considered a necessity. Summary More than 18 million people worldwide are suffering from Alzheimer disease, the most common dementing disorder in humans. Transgenic lines have been created in order to understand the underlying mechanisms of Alzheimer disease and to find a cure. None of the available models completely recapitulates the characteristics of human pathology, but they provide valuable information on different pathogenic pathways involved. New therapeutic approaches and improvement of current strategies can be obtained from the use of Alzheimer animal models.

Localization of cholesterol, amyloid and glia in Alzheimer’s disease transgenic mouse brain tissue using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and immunofluorescence imaging

The spatial distributions of lipids, amyloid-beta deposits, markers of neurons and glial cells were imaged, at submicrometer lateral resolution, in brain structures of a mouse model of Alzheimer’s disease using a new methodology that combines time-of-flight secondary ion mass spectrometry (ToF-SIMS) and confocal fluorescence microscopy. The technology, which enabled us to simultaneously image the lipid and glial cell distributions in Tg2576 mouse brain structures, revealed micrometer-sized cholesterol accumulations in hippocampal regions undergoing amyloid-beta deposition. Such cholesterol granules were either associated with individual amyloid deposits or spread over entire regions undergoing amyloidogenesis. Subsequent immunohistochemical analysis of the same brain regions showed increased microglial and astrocytic immunoreactivity associated with the amyloid deposits, as expected from previous studies, but did not reveal any particular astrocytic or microglial feature correlated with cholesterol granulation. However, dystrophic neurites as well as presynaptic vesicles presented a distribution similar to that of cholesterol granules in regions undergoing amyloid-beta accumulation, thus indicating that these neuronal endpoints may retain cholesterol in areas with lesions. In conclusion, the present study provides evidence for an altered cholesterol distribution near amyloid deposits that would have been missed by several other lipid analysis methods, and opens for the possibility to study in detail the putative liaison between lipid environment and protein structure and function in Alzheimer’s disease.

H1N1 influenza virus induces narcolepsy-like sleep disruption and targets sleep–wake regulatory neurons in mice

Significance Influenza A virus infections are risk factors for narcolepsy, a disease in which autoimmunity has been implicated. We tested experimentally whether influenza virus infections could be causally related to narcolepsy. We found that mice infected with a H1N1 influenza A virus strain developed over time sleep–wake changes described in murine models of narcolepsy and narcolepsy patients. In the brain, the virus infected orexin/hypocretin-producing neurons, which are destroyed in human narcolepsy, and other cells in the distributed sleep–wake-regulating neuronal network. The findings, obtained in mice lacking an adaptive autoimmune response, thus provide new avenues for research on infection-related mechanisms in narcolepsy. An increased incidence in the sleep-disorder narcolepsy has been associated with the 2009–2010 pandemic of H1N1 influenza virus in China and with mass vaccination campaigns against influenza during the pandemic in Finland and Sweden. Pathogenetic mechanisms of narcolepsy have so far mainly focused on autoimmunity. We here tested an alternative working hypothesis involving a direct role of influenza virus infection in the pathogenesis of narcolepsy in susceptible subjects. We show that infection with H1N1 influenza virus in mice that lack B and T cells (Recombinant activating gene 1-deficient mice) can lead to narcoleptic-like sleep–wake fragmentation and sleep structure alterations. Interestingly, the infection targeted brainstem and hypothalamic neurons, including orexin/hypocretin-producing neurons that regulate sleep–wake stability and are affected in narcolepsy. Because changes occurred in the absence of adaptive autoimmune responses, the findings show that brain infections with H1N1 virus have the potential to cause per se narcoleptic-like sleep disruption.

Effects of spatial and cognitive enrichment on activity pattern and learning performance in three strains of mice in the IntelliMaze.

The IntelliMaze allows automated behavioral analysis of group housed laboratory mice while individually assigned protocols can be applied concomitantly for different operant conditioning components. Here we evaluate the effect of additional component availability (enrichment) on behavioral and cognitive performance of mice in the IntelliCage, by focusing on aspects that had previously been found to consistently differ between three strains, in four European laboratories. Enrichment decreased the activity level in the IntelliCages and enhanced spatial learning performance. However, it did not alter strain differences, except for activity during the initial experimental phase. Our results from non-enriched IntelliCages proved consistent between laboratories, but overall laboratory-consistency for data collected using different IntelliCage set-ups, did not hold for activity levels during the initial adaptation phase. Our results suggest that the multiple conditioning in spatially and cognitively enriched environments are feasible without affecting external validity for a specific task, provided animals have adapted to such an IntelliMaze.

Simultaneous imaging of amyloid-β and lipids in brain tissue using antibody-coupled liposomes and time-of-flight secondary ion mass spectrometry

The spatial localization of amyloid-β peptide deposits, the major component of senile plaques in Alzheimers disease (AD), was mapped in transgenic AD mouse brains using time-of-flight secondary ion mass spectrometry (ToF-SIMS), simultaneously with several endogenous molecules that cannot be mapped using conventional immunohistochemistry imaging, including phospholipids, cholesterol and sulfatides. Whereas the endogenous lipids were detected directly, the amyloid-β deposits, which cannot be detected as intact entities with ToF-SIMS because of extensive ion-induced fragmentation, were identified by specific binding of deuterated liposomes to antibodies directed against amyloid-β. Comparative investigation of the amyloid-β deposits using conventional immunohistochemistry and fluorescence microscopy suggests similar sensitivity but a more surface-confined identification due to the shallow penetration depth of the ToF-SIMS signal. The recorded ToF-SIMS images thus display the localization of lipids and amyloid-β in a narrow (~10 nm) two-dimensional plane at the tissue surface. As compared to a frozen nontreated tissue sample, the liposome preparation protocol generally increased the signal intensity of endogenous lipids, likely caused by matrix effects associated with the removal of salts, but no severe effects on the tissue integrity and the spatial distribution of lipids were observed with ToF-SIMS or scanning electron microscopy (SEM). This method may provide an important extension to conventional tissue imaging techniques to investigate the complex interplay of different kinds of molecules in neurodegenerative diseases, in the same specimen. However, limitations in target accessibility of the liposomes as well as unspecific binding need further consideration.

Cognitive impairment in the Tg6590 transgenic rat model of Alzheimer’s disease

Recently, interest in the rat as an animal model of Alzheimer’s disease (AD) has been growing. We have previously described the Tg6590 transgenic rat line expressing the amyloid precursor protein containing the Swedish AD mutation (K670M/N671L) that shows early stages of Aβ deposition, predominantly in cerebrovascular blood vessels, after 15 months of age. Here we show that by the age of 9 months, that is long before the appearance of Aβ deposits, the Tg6590 rats exhibit deficits in the Morris water maze spatial navigation task and altered spontaneous behaviour in the open‐field test. The levels of soluble Aβ were elevated both in the hippocampus and cortex of transgenic animals. Magnetic resonance imaging showed no major changes in the brains of transgenic animals, although they tended to have enlarged lateral ventricles when compared to control animals. The Tg6590 transgenic rat line should prove a suitable model of early AD for advanced studies including serial cerebrospinal fluid sampling, electrophysiology, neuroimaging or complex behavioural testing.